Modifications of transfer RNA (tRNA) molecules occur after transcription and constitute an essential step to promote cellular fitness and viability. Of interest is the m1G37 modification, which takes place at almost all tRNA molecules that have the G37 base immediately adjacent to the 3'side of the anticodon sequence. The m1G37 modification is important for maintaining the tRNA reading frame specificity on the ribosome during protein synthesis. Elimination of this modification increases ribosomal errors in protein synthesis and elevates frequencies of frame shifts. The enzyme that catalyzes the m1G37 modification is tRNA(m1G37) methyl transferase, which transfers the methyl group of S-adenosyl methionine (AdoMet) to the N1 position of G37 in tRNA. The bacterial enzyme, known as TrmD (encoded by the trmD gene), is essential for growth in E. coli, Salmonella typhimurium, and Streptococcus pneumoniae. An unexpected recent finding is that while TrmD is highly conserved among bacterial species, it shares little sequence or structural homology with its eukaryotic and archaeal counterpart (known as Trm5, encoded by the trm5 gene). This establishes TrmD and Trm5 as a pair of analogous enzymes that use distinct structural folds to catalyze the same reaction to synthesize the growth-dependent m1G37 in tRNA. The separation of TrmD and Trm5 along the split of bacteria from eukarya- archaea thus raises the medically relevant and attractive prospect of selective targeting of the bacterial TrmD. This proposal is aimed at providing a strong biochemical and molecular foundation that is necessary for the success of such drug targeting. Two aims of the project are to determine the molecular and structural basis of TrmD and Trm5 for their recognition of AdoMet and tRNA and the dynamic recognition process that leads to catalysis. The third aim will determine the role of the m1G37 modification on the ribosome during the decoding process. Together, these aims combine the strengths and interests of two presently separate fields (enzymology of tRNA modification, and ribosome structure and function) to address key issues that will have long-term impact on human health and bio-defense against bacterial pathogens. PUBLIC HEALTH RELEVANCE: The cellular protein synthesis machinery provides the basis for translating genetic information stored in nucleic acids to functional proteins. This machinery depends on extensive tRNA-ribosome interactions to ensure the fidelity of protein synthesis. The m1G37 modification of tRNA is a key determinant of this fidelity and is essential for growth for several bacterial pathogens. However, the molecular and biochemical basis for how the m1G37 modification is synthesized and how it functions on the ribosome is poorly understood. Preliminary studies show that the enzyme that catalyzes the m1G37 synthesis in bacteria was of a different structural origin from its counterpart in eukaryotes and in archaea. This proposal is aimed at elucidating the molecular basis for the enzymatic synthesis of m1G37 and the differences between the bacterial and eukaryotic/archaeal enzymes. Information to be gained from this proposal will provide important insights to build a solid foundation for selective inhibition of the bacterial enzyme so as to improve human health care.